Environmental threat, physical exertion or injury and psychological strain all lead to the initiation of the sympatho-adrenal fight or flight stress response. Neuroendocrine adrenal medullary chromaffin cells receive excitatory synaptic input from the sympathetic splanchnic nerve. Splanchnic activation causes adrenal chromaffin cells to release catecholamines as well as a diverse array of neuro- and vaso-active peptide transmitters into the circulation. Different levels of stress result in the differential release of catecholamine versus peptide transmitters to formulate the appropriate physiological response. All secreted hormones, catecholamines as well as multiple species of peptide transmitters, are co-packaged in the same secretory granules. Thus, differential hormone release is regulated at a step after granule fusion. Under basal chromaffin cell excitation, set by the sympathetic tone, selective release of freely-soluble catecholamine occurs through a transient fusion event, characterized by a narrow, structured exocytic fusion pore between the granule lumen and the extracellular space. Under sympathetic tone, selective and modest catecholamine release plays an important role in the rest and digest metabolic status of energy storage, regulating homeostatic physiological functions including pancreatic insulin secretion, increased blood flow to the viscera and maintenance of basal cardiac activity. In response to stress, increased chromaffin cell stimulation modulates the mode of secretory granule fusion, leading to the expansion of the exocytic fusion pore to maximize catecholamine release and facilitate exocytosis of the co-packaged adrenal peptide transmitters. Elevated serum catecholamine levels, in combination with adrenal-derived peptide transmitters, are core effectors of the sympathetic fight or flight stress response. Together they regulate multiple processes that prepare for defense or escape, including generalized analgesia (enkephalin), increased cardiac output (elevated catecholamines), blood flow to skeletal muscle (atrial natriuretic factor, Neuropeptide Y) and blood glucose (pancreastatin). Thus, regulated expansion of the secretory fusion pore represents a key element of the acute stress response. It is our overall goal to understand the molecular mechanism responsible for pore expansion. We summarize previous work and provide preliminary data to formulate 3 specific aims to test an activity-dependent dynamin I de- phosphorylation event, subsequent recruitment of a multimeric pore-expansion complex, and requirement for myosin motor activity in the regulation of fusion pore expansion. In the execution of these aims, we employ state of the art electrophysiological, electrochemical and quantitative fluorescence microscopy as well as a newly-developed silicon nanowire-based field effect transistor (SiNW-FET) biosensor to measure specific peptide release. The data obtained will provide a molecular understanding of the key regulators of the acute sympatho-adrenal stress response as well as pathologies resulting from its improper regulation. PUBLIC HEALTH RELEVANCE: Environmental or physical threat increases firing of the autonomic sympathetic nervous system to activate the acute fight or flight stress response. Through an as-of-yet poorly understood process, different sympathetic activity levels result in unique hormonal profiles to elicit appropriate physiological responses. This proposal will test a molecular signaling cascade for the specific activity-dependent differential release of hormone transmitters into the circulation and thus provide the mechanistic basis for the sympatho-adrenal acute stress response.